Introduction

Tick-borne diseases are an important cause of morbidity and mortality in dogs worldwide with the brown dog tick, Rhipicephalus (R.) sanguineus, implicated as a vector of several disease agents including A. platys, B. canis vogeli, B. gibsoni, E. canis, spotted fever group (SFG) Rickettsia spp. and H. canis [1], [2], [3], [4]. Although there is some information on tick-borne infections of dogs in the Caribbean, each island has a unique immigration history and vector ecology and additional studies are needed to more fully understand infections in the region. Anecdotal reports suggested canine monocytic ehrlichiosis (CME) caused by E. canis occurs on Aruba, Puerto Rico, the Virgin Islands and Netherlands Antilles [5], [6], [7]. In early PCR studies, no DNA of SFG rickettsia was found in R. sanguineus, the most common tick of dogs in the Caribbean, from the islands of St Kitts (n = 52) [8] and Martinique (n = 11) [9]. But in a later study on St Kitts, Kelly et al. [10] found DNA of SFG rickettsiae in 6% of 143 R. sanguineus. They also found 15% had DNA of Ehrlichia spp., but none contained Anaplasma DNA. A PCR study on 73 dogs from Grenada showed them to be infected with A. platys (19%), B. canis (7%), Bartonella spp. (1%), E. canis (25%) and H. canis (7%) [11] while a study on 348 dogs from Trinidad showed dogs infected with E. canis (14%) and B. canis vogeli (7%) [12]. In a serosurvey of dogs from the Turks and Caicos Islands, 71% of feral dogs were positive for E. canis infection compared to 18% of pet dogs presenting to a veterinary clinic [13]. In the most recent study from the Caribbean, 13 thrombocytopenic dogs from St. Kitts were negative for A. platys and Babesia organisms by PCR [14].

To provide further information on tick-borne pathogens, we studied a large number of dogs from St Kitts to more precisely determine the prevalence of infections with tick-borne agents and obtain clinical data on infected animals.

Materials and Methods

All work in this study was reviewed and approved by the Institutional Animal Care and Use Committee of the Ross University School of Veterinary Medicine.

DNA Extraction and Quantitative PCR

The High-Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) was used to extract total nucleic acid from canine whole blood as we previously described [15]. For each sample, 200 µl EDTA-whole blood was mixed with an equal volume of binding buffer [6 M guanidine-HCl, 10 mM urea, 20% (v/v) Triton X-100, 10 mM Tris-HCl, pH 4.4], and DNA samples were eluted in 150 µl elution buffer.

Quantitative PCR for E. canis 16S rRNA was performed on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystem, Foster City, CA, USA) as described previously [16]. Quantitative fluorescence resonance energy transfer (FRET)-PCRs for 16S or 18S rRNA gene of Babesia spp., Anaplasma spp. and Hepatozoon spp. were performed on a Roche LightCycler 2.0 PCR Instrument. Following the completion of FRET-PCR, the melting curve analysis for probes annealing to the PCR products was determined by monitoring the fluorescence from 37°C to 85°C with a temperature transition rate of 0.2°C per second [15]. The fluorescence ratio F4/F1 was analyzed, and the first derivatives of F4/F1 were evaluated to determine the probe melting temperature (T_m)_.

The forward and reverse primers and the LCRed 640 probe used for Babesia qPCR had sequences common to all Babesia spp. strains [15] while the fluorescein probe (5′-GACCC AAAAT CTCAC CAGAG TAACA ATTGG-6-FAM-3′) had two nucleotide mismatches to B. gibsoni and three mismatches to B. canis vogeli (Figure 1). The Anaplasma qPCR primers (forward primer: 5′-GGT CGC AAG ACT AAA ACT CAA AGG AAT TGA CG-3′; reverse primer: 5′-TTA ACC CAA CAT CTC ACG ACA CGA GCT-3′) were designed to amplify portions of the 16S rRNA gene of both Ehrlichia spp. and Anaplasma spp., and specific probes were used to detect only Anaplasma spp. Two FRET probes [anchor probe: 5′-ACG CGA AAA ACC TTA CCA CTC CTT GAC–(6-FAM)-3′; quencher probe: 5′-(Bodipy 630/650)-TGG AGA TTA GAT CCT TCT TAA CGG AAG GG–(Phosphate)-3′] had identical sequences to A. phagocytophilum (AY055469, CP000235) and two nucleotide mismatches with A. platys (M82801, EF139459), but 8 with E. ewingii (M73227, U96436), 9 with E. canis (GU810149, EU178797) and 10 with E. chaffeensis (AF147752, CP000236). A nucleotide fragment representing part of the 16S rRNA gene of A. phagocytopilum was synthesized and inserted in the pIDTSMART cloningVector (Integrated DNA Technologies, Coralville, IA, USA). The plasmid was linearized with HindIII (Promega, Madison, WI, USA) and the restriction enzyme inactivated at 65°C for 20 min. DNA was quantified by the PicoGreen DNA fluorescence assay (Molecular Probes, Eugene, OR, USA) for preparation of quantitative standards in TE buffer.

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Figure 1. Sequencing results and melting curves of the Babesia spp.

FRET-PCR. The upstream and downstream primers and the LCRed 640 probe of Babesia spp. FRET PCR are identical to the sequences of all Babesia spp. strains [15]. The fluorescein probe (5′-GACCC AAAAT CTCAC CAGAG TAACA ATTGG-6-FAM-3′) has two nucleotide mismatches to B. gibsoni (A, C; only a portion of the sequencing results were shown here), and three mismatches to B. canis canis and B. canis vogeli (B, C). Melting curves show the distinct T_m differences between B. gibsoni (∼63.5°C; D), and B. canis vogeli (∼57.0°C; E) as well as dual peaks indicating co-infections of B. gibsoni and B. canis vogeli (F).

https://doi.org/10.1371/journal.pone.0053450.g001

The PCR for Hepatozoon spp. was performed as previously described [17]. High-resolution melting curve analysis was used to discriminate H. canis (T_m as ∼56°C) and H. americanum (T_m as ∼65°C).

The sensitivity and specificity of qPCRs for the detection of E. canis, Babesia spp. and Hepatozoon spp. were determined as described previously [15], [16], [17]. The size of the amplification products of the qPCRs were verified by gel electrophoresis and the amplicons were purified for DNA sequencing with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Nucleotide sequencing was performed at the Genomic Sequencing Laboratory (Davis Sequencing, Davis, CA) using forward and antisense primers described here or in previous publications [15], [16], [17]. The specificity of Anaplasma qPCR was confirmed by DNA sequencing as well as testing on DNAs of A. platys, A. phagocytophilum, E. canis, E. ewingii and E. chafeensis. The Anaplasma PCR enabled the detection of 2 copies of 16S rRNA per PCR reaction which is equivalent to 150 copies of target DNA per ml of whole blood.

Results

Prevalence

The most common tick-borne agent in our study was E. canis with 24% (88/372) of the dogs having PCR and/or serological evidence of infection (Table 1). Nineteen-percent (69/372) were positive by PCR with the prevalence being significantly lower in dogs presenting to the VTH (11%; 31/279) compared to those attending the VIDA clinics (41%; 38/93) (P = 0.001). The average copy number in the E. canis PCR for dogs attending the VIDA clinics (148,362 copies of 16S rRNA/ml whole blood; n = 38) was significantly higher than that for the dogs presenting to the VTH (344 copies of 16S rRNA/ml whole blood; n = 31) (P = 0.02). Of the 125 owners of dogs presenting to the VTH that were questioned on tick control, most (75%; 94/125) reported the use of tick collars or spot-on preparations that local experience suggests are effective on the island. Although similar data was not available for owners of dogs presented to the free VIDA clinics, experience suggests that these dogs are commonly infested with ticks as the owners are unable to afford commercial tick control products.

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Table 1. Prevalences of infections with tick-borne disease agents and numbers of dogs on which full data was available and their health status.

https://doi.org/10.1371/journal.pone.0053450.t001

The immunochromatography test results showed 19% (33/170) of the dogs tested from the VTH had antibodies reactive with E. canis. In the dogs that were tested by both PCR and the immunochromatography test, agreement between the two tests was present in 87% (148/170) of patients with both tests being negative for 84% (142/170) of the dogs and both tests being positive for 4% (6/170). Only the immunochromatography test was positive in 11% (19/170) of dogs and only the PCR was positive in 2% (3/170).

Our PCR study on Babesia spp. showed infections with the organisms were common (24%; 90/372) with the percentage of infected dogs that presented to the VTH (25%; 70/279) being similar to that for dogs attending the VIDA clinic (21%; 20/93). The fluorescent probe with its different level of mismatches with Babesia spp. and subspecies gave distinct T_m differences (Figure 1) which enabled us to determine and confirm by sequencing that there are two Babesia spp. on St Kitts, mainly B. canis vogeli (48%; 43/90) and B. gibsoni (40%; 36/90). Two dogs had DNA of both species as evidenced by dual melting point curves and 9 had unique melting temperatures which could not be accurately identified to the species level due to the limited length of the PCR amplicon available for sequencing. We obtained partial sequences (221 nucleotides of the 18S rRNA gene) for 27 of the dogs positive by PCR for Babesia spp. and, using the BLAST program (www.ncbi.nim.nih.gov/blast/), we found that the sequences of the B. canis vogeli (n = 12) from St Kitts were identical to one another, and to most of those reported from around the world including from Venezuela (DQ297390), Brazil (AY371194), Germany (AY371197) and the USA (AY072925). The sequences we obtained for the B. gibsoni from St. Kitts (n = 15) were also identical to one another, and to the vast majority of those reported from around the world, for example from widely disparate areas such as Australia (AY102164), Japan (AB118032), the USA (EU084677) and Germany (AF175300).

Immunochromatography tests for antibody to A. phagocytophilum/A. platys were performed on 157 blood samples with 17 giving positive reactions. The qPCR showed A. platys in 4 of these blood samples (28%) with a T_m of 59.6°C which was substantially lower than the 69.6°C T_m for the A. phagocytophilum sequence-containing plasmid standard. The presence of A. platys in the 4 positive amplicons was further confirmed by DNA sequence analysis.

Six percent (15/266) of the dogs tested by PCR had evidence of infection with Hepatozoon spp. Melting curve analysis indicated all were H. canis with a T_m of 58°C and this was confirmed by DNA sequencing of 4 amplicons.

Exposure of dogs to multiple agents was relatively common with 14% (51/372) having evidence of dual exposures. Those to E. canis and B. canis vogeli (35%; 14/51) and E. canis and B. gibsoni (22%; 11/51) were most common, while dual exposures to A. platys and E. canis (8%; 4/51) and B. canis vogeli and H. canis (2%; 1/51) were the least common. No dual exposures to H. canis and A. platys were seen while triple exposures to E. canis, B. canis vogeli and A. platys occurred in 4 dogs.

Discussion

Our study showed dogs on the island of St. Kitts in the Caribbean are commonly exposed to tick-borne organisms. Exposure to E. canis was the most common (24%) and this is consistent with reports that CME is very common on some other Caribbean islands from which data is available, Grenada (25%) [11], Turks and Caicos Islands (71%) [13] and Trinidad (14%) [12], and also around the world [19], [20], [21]. The vector of E. canis is the brown dog tick, R. sanguineus, which occurs commonly on St Kitts and is effectively the only tick found on dogs on the island [8], [14]. The significant difference in the prevalence of E. canis exposure in dogs presenting to the VTH and those presenting to VIDA was probably due to decreased infections resulting from more effective control of ticks in the former group. The higher copy numbers in the dogs presenting to VIDA might represent more acute infections being seen in these dogs [16] or it might result from different strains of E. canis circulating on the island [22].

We had full data on 24 of the 28 dogs which had immunochromatography test and/or PCR evidence of E. canis infection and no indication of other tick-borne diseases (Table 1). The finding of evidence of an E. canis infection in 48% (12/25) of these dogs was unexpected and occurred only because the dogs were having routine health checks which included testing for heartworms, the test kit for which includes the immunochromatography test for antibodies to E. canis. Although without signs of a tick-borne disease, five of the 12 dogs (42%) had laboratory abnormalities consistent with E. canis infection including thrombocytopenia, anemia, leukopenia and hyperglobulinemia [19], [23]. These abnormalities can affect the outcome of routine surgeries and testing for E. canis in dogs from the region should be considered before such procedures are performed.

The dogs found positive for E. canis in our study were treated according to the AVMA Consensus Statement on ehrlichial disease of small animals which was based on information available before its publication in 2002. It recommends dogs be given doxycycline if they are seropositive and/or PCR positive and there are compatible clinical and laboratory abnormalities [18]. Unfortunately, the best course of action for the 7 dogs we found that were immunochromatography test positive but normal clinically and by laboratory testing is not as straightforward. The AVMA Consensus Statement lists points for and against treating such dogs and the consensus is that treatment should be discussed with the owner and a decision made on the management course which is in the best interests of the dog [18]. A major consideration is that treatment of such animals will prevent them from progressing to the chronic phase of the disease (see below) where treatment is often unsuccessful and mortality is high [24].

In only 52% of cases (13/25) did clinicians specifically order the immunochromatography test for E. canis. These tests were requested for a dog with clinical signs of a tick-borne disease, two dogs presenting because of trauma which had inappropriately low platelet counts suspected to be due to E. canis infection, and 10 dogs being rechecked after appropriate treatment with doxycycline for E. canis infections [18]. The criteria for successful treatment are very clear in the AVMA Consensus Statement, mainly that dogs should become clinically normal, laboratory abnormalities resolve and the PCR and serology become negative [18]. Determining the success of treatment in practice, however, is generally not easy as laboratory abnormalities might take time to return to normal after treatment with thrombocytopenia usually taking 14 days and hyperglobulinemia taking up to nine months to normalize [18]. Further, although most dogs become seronegative by six to nine months after treatment, titers can remain elevated for years in some dogs [25], [26]. Also, although PCR is usually negative within two weeks of treatment [16] [27], [22], in some dogs it can remain intermittently positive after treatment [28]–[29].

All the dogs in our study failed to meet the criteria for successful treatment although three dogs with normal laboratory work were sampled seven to eight months post treatment, perhaps before the antibody titers had declined to non-detectable levels. Although immunochromatography tests are specific, sensitive and quick and simple to perform, they record any titer over about 1/100 as positive and it is not possible to determine if serum titers are declining [30], [31]. It should be noted, however, that the above dogs had all been treated two to three times previously, as had four of the six dogs that remained seropositive and still had laboratory abnormalities. Five of the latter dogs had been treated over nine months earlier and there should have been time for laboratory abnormalities and antibody titers to resolve.

Treatment failures with doxycycline have been reported in experimentally [28], [29], [32], [33], [34], [35] and naturally infected dogs [25], [22], [33]. There have been no carefully controlled longitudinal studies of dogs treated with doxycycline to determine the reasons for the apparent treatment failures. These have been summarized [19] and include antibiotic resistance, doxycycline administration problems, sequestration of E. canis and aberrant immune responses. Antibiotic resistance seems unlikely as in vitro susceptibility studies have shown that doxycycline has a bactericidal effect on E. canis at very low concentrations [36] and the susceptibility of the organism to the drug has been supported in many experimental infection studies [16], [27] [37]. A more likely reason for treatment failures is reinfection with E. canis as there appears to be no protective immune response in dogs after exposure and dogs can be reinfected following spontaneous or treatment clearance of infections [37], [38]. It is, then, very important that tick control measures are introduced to prevent reinfection post treatment. Although this is the policy of the VTH, owner compliance is not always complete so the authors regard this to be the most likely cause of the treatment failures we observed. This was most likely the case for the one recheck we found to be actively infected, that is PCR and immunochromatography test positive for E. canis and with laboratory abnormalities.

Our immunochromatography and PCR test results for E. canis were in agreement in a large percentage of dogs, supporting earlier reports of the sensitivity and specificity of immunochromatography tests [30], [31]. Similarly, our finding that PCR and immunochromatography tests were not always in agreement has also been reported previously, for example in dogs from Hong Kong [20], Colombia [21] and Grenada [11]. In these reports, 0% to 5% of dogs were immunochromatography test negative and PCR positive, which is similar to our findings of 2%. Similarly, about 17% (3% to 42%) were PCR negative but immunochromatography test positive, which is also comparable to our findings of 11%. These anomalous results probably arise because dogs are in different stages of infection.

Early experimental studies showed infections with E. canis have three phases, the acute, subclinical and chronic phases [38], [39], [40]. Dogs experimentally infected with E. canis usually become PCR positive within 4–10 days [16], [33], [41] but can take up to 28 days to develop reactive antibodies [42]. Therefore, animals that are PCR positive and immunochromatography test negative are considered to be in the acute phase of CME. Although experimental infections of dogs with E. canis cause clinical signs and laboratory changes in all dogs [16], [28], [32], [37], it is commonly stated that naturally acquired acute infections may be subclinical [18], [19], [23]. This seems to be based on wide experience of finding dogs infected with E. canis but with no history of compatible clinical signs. Although our numbers were small, the data supports this observation with 3 of the 4 dogs that appeared to be acutely infected, that is PCR positive and immunochromatography test negative, being clinically normal.

After the acute phase, dogs enter the subclinical phase of infection where they remain PCR positive and immunochromatography test positive [34], [43]. Although apparently healthy, they commonly have laboratory abnormalities, in particular thrombocytopenia (86%), anemia (57%), hyperglobulinemia (39%) and leukopenia (31%), singly or in combinations [18], [19], [43], [44]. In our study we found four dogs with full data in this stage of the disease. All were apparently healthy with only one having a laboratory abnormality which provides further substantiation that many natural E. canis infections are subclinical [24], [51]. As in acute infections, deciding on treatment for animals with no laboratory abnormalities is difficult.

Following the subclinical phase of the disease some dogs will have spontaneous resolution of infections [34], [35], [37]. Such dogs have no history of treatment but become PCR negative with laboratory abnormalities and antibody titers declining slowly over six to nine months. In our study, 24% (6/25) of the dogs with evidence of E. canis infection were immunochromatography test positive, PCR negative and with no clinical or laboratory abnormalities. These dogs could, then, have been undergoing spontaneous resolution of their infections. This is consistent with the proportion of dogs described to have spontaneous resolution in experimental infections (30–70%) [34], [35], [37], and supports a previous report that spontaneous recovery also occurs in naturally infected dogs [45].

In rare instances, following the subclinical phase of the disease, a severe and life-threatening chronic phase of the disease may develop. In this phase, dogs are usually seropositive and have clinical signs including anorexia, weight loss, emaciation, bleeding tendencies and severe pancytopenia with hypoplastic bone marrow [24], [39]. Our study confirmed that the chronic phase of the disease is uncommon as, from all the dogs on which we had full data, we could only identify 3 (7%; 3/42) that were probably in this phase of the disease. One was seropositive for E. canis and 2 were immunochromatography and PCR test positive and coinfected with B. canis vogeli. We found only one report describing the PCR status of dogs in the chronic phase of the disease and this showed most (13/19) had bone marrow positive by PCR for E. canis [24]. Our finding of 2/3 dogs with positive PCRs of whole blood suggests either sample can be used for confirming E. canis infection and further demonstrates that detecting E. canis in pancytopenic dogs in the chronic phase of the disease can be difficult.

Canine babesiosis is caused by the small B. gibsoni and three strains of the large B. canis namely B. canis canis, B.canis vogeli and B. canis rossi. The results of a previous study on St Kitts indicated there was no babesiosis on the island as DNA of Babesia spp. was not found in 13 dogs from the island [14]. The primers used in that study were developed to detect US strains [46] but should have detected the Babesia we found as our sequencing data showed them to be closely related to strains in the US and others around the world. A more likely explanation for the inability to find Babesia sp. in the above study is the fact that the only dogs tested were those that were apparently healthy, thrombocytopenic and seronegative for E. canis. We found that only a small percentage of such dogs infected with Babesia spp. have thrombocytopenia (11%; 2/17) suggesting the above study's sample group may not have been ideal for detecting Babesia spp.

We failed to identify B. canis canis and B. canis rossi on the island probably because their vectors, Dermacentor spp. [47] and Haemaphysalis elliptica [48], respectively, are not present on St Kitts. The brown dog tick, R. sanguineus, is the vector of B. canis vogeli [47] and is a very common tick on dogs on the island [8], [14]. It is not surprising that this was the principal species we encountered causing 48% (43/90) of the Babesia spp. infections. Babesia canis vogeli is found widely in the tropical and subtropical regions of most continents and has been reported from the Caribbean previously with 7% of dogs in both Grenada [11] and Trinidad [12] being infected. Organisms were not seen in 187 blood smears from dogs on the Turks and Caicos Islands [13] but our laboratory also did not report parasites in routine blood films, even though we had a high prevalence of infection by PCR. In the US, prevalences vary from 4–59% with most infections being subclinical and most infected dogs being subclinical carriers [49]. This was also the situation on St Kitts where we found only a single case (2%) of B. canis vogeli causing acute clinical signs in the 43 infected dogs. The infections in the remaining dogs were incidental findings when the dogs presented for routine health checks or for other conditions and only occasionally were mild laboratory changes seen.

B. gibsoni is found in northern Africa, southern Asia, Australia, Europe and the USA [47]. We now report it from the Caribbean. It would appear, however, that the organism is not widely distributed in the region as recent studies on Grenada and Trinidad failed to identify B. gibsoni [11], [12]. There are a number of suspected vectors of B. gibsoni, mainly R. sanguineus, H. longicornis, H. leachii, H. bispinosa and D. variabilis [49]. The fact that the only tick on this list found on St Kitts is R. sanguineus provides further evidence for it being a significant vector of B. gibsoni. High prevalence of infection in pit bulls in the US has led to suggestions that this breed is more susceptible and fighting might be a means of transmission of B. gibsoni [50], [51]. This appeared not to be the case in our study, however, as pit bulls and their crosses comprised 6% of the dogs we studied yet they comprised only 4% (2/46) of the dogs we found infected with B. gibsoni.

Our sequencing data showed the Babesia spp. we found on St Kitts were identical to one another and those from the US and around the world and did not enable us to determine their geographic origin. We do know, however, that students from the US commonly bring their dogs with them when attending universities on the island. It is likely that some of these dogs could have been subclinically infected and a source of infection for the R. sanguineus vectors and naïve dogs on the island. Recently, new Babesia spp. of dogs have been described in the United States including Babesia sp. (Coco) [52] and B. conradae [53], and animal health authorities on Caribbean islands might be advised to request PCR screening of visiting dog for Babesia to prevent introductions of further species. We were unable to identify the Babesia sp. in 9 of the dogs we studied although the sequence data we obtained allowed us to determine they were not the above organisms or Theileria equi and B. caballi, species known to infect horses on the island and dogs in Europe [54], [55]. Further work is underway in our laboratory to further characterize these Babesia spp.

Babesia gibsoni is generally regarded as a pathogenic species causing acute signs including fever, pallor, anorexia and weight loss [56]. There is also a chronic form that is seen frequently in the US [49] and has the above signs and splenomegaly, hepatomegaly and lymphadenopathy. Asymptomatic infections have also been reported [46] [50] [55] and this was our experience on St Kitts with over half of the dogs for which we had full data having no clinical or laboratory abnormalities. When they were present, they were mild and consistent with those previously reported [56].

Although the immunochromatography test detects antibodies to both A. phagocytophilum and A. platys, the vectors for the former, Ixodes spp., is not present on St Kitts and this, together with our T_m and sequencing results, demonstrated that A. platys was the organism we detected in our study dogs. The US strains of A. platys, the agent of infectious canine thrombocytopenia, generally cause subclinical infections in dogs while those from elsewhere are more likely to cause clinical signs including fever, lymphadenopathy and bleeding disorders [57], [58], [59]. The A. platys we found on St Kitts seems to be most similar to that in the US. Most of the dogs we found with evidence of infection with the organism were apparently healthy with no laboratory abnormalities. This included three dogs that were PCR positive and thus most likely with active infections.

We detected H. canis infections in 4% of the dogs sampled but, as is the case elsewhere, the organism appears to be of low pathogenicity [3]. Most of the infected dogs on St Kitts had no clinical or laboratory signs. Laboratory abnormalities were seen in dogs coinfected with E. canis but these were most likely due to the E. canis infection.

In conclusion, our data provides further evidence that dogs in the Caribbean are commonly infected with tick-borne agents and veterinarians treating these animals should have a high index of suspicion of infection. Further, dogs from the Caribbean are commonly subclinical carriers of tick-borne organisms and animal health workers dealing with the importation of these dogs into non-endemic areas should consider appropriate testing and quarantine.

Acknowledgments

The authors sincerely appreciate Dr. Rosina (Tammi) Krecek for the fruitful discussions and Ms. Alana Hefter for the data entry.